EmbA is an essential arabinosyltransferase in Mycobacterium tuberculosis

Abstract

The Emb proteins (EmbA, EmbB, EmbC) are mycobacterial arabinosyltransferases involved in the biogenesis of the mycobacterial cell wall. EmbA and EmbB are predicted to work in unison as a heterodimer. EmbA and EmbB are involved in the formation of the crucial terminal hexaarabinoside motif [Arabeta(1-->2)Araalpha(1-->5)] [Arabeta(1-->2)Araalpha(1-->3)]Araalpha(1-->5)Araalpha1-->(Ara(6)) in the cell wall polysaccharide arabinogalactan. Studies conducted in Mycobacterium smegmatis revealed that mutants with disruptions in embA or embB are viable, although the growth rate was affected. In contrast, we demonstrate here that embA is an essential gene in Mycobacterium tuberculosis, since a deletion of the chromosomal gene could only be achieved when a second functional copy was provided on an integrated vector. Complementation of an embA mutant of M. smegmatis by M. tuberculosis embA confirmed that it encodes a functional arabinosyltransferase. We identified a promoter for M. tuberculosis embA located immediately upstream of the gene, indicating that it is expressed independently from the upstream gene, embC. Promoter activity from P(embA)((Mtb)) was sevenfold lower when assayed in M. smegmatis compared to M. tuberculosis, indicating that the latter is not a good host for genetic analysis of M. tuberculosis embA expression. P(embA)((Mtb)) activity remained constant throughout growth phases and after stress treatment, although it was reduced during hypoxia-induced non-replicating persistence. Ethambutol exposure had no effect on P(embA)((Mtb)) activity. These data demonstrate that M. tuberculosis embA encodes a functional arabinosyltransferase which is constitutively expressed and plays a critical role in M. tuberculosis.

Fig. 1.

Demonstration of essentiality of embA in M. tuberculosis. (a) The genomic organization of the embA region is shown. The regions marked by black bars were amplified and cloned into p2NIL to generate the delivery vector pEMPTY16. The promoter region assayed for activity is indicated by an arrow. (b) and (c) Southern analysis of recombinant strains. (b) The maps of the wild-type and deletion mutant are shown, with the expected sizes. BamHI sites are indicated by symbols (0) and the probe is shown as a solid bar. (c) No deletion DCOs were isolated in the wild-type background. PCR was used to screen for potential deletion alleles in the merodiploid background and four potential del-int strains were identified. Genomic DNA from these four strains was digested with BamHI and hybridized to the probe. The expected sizes for the wild-type and integrated (int) copies of embA are given. Lanes: 1–4, genomic DNA from four del-int strains (deletion DCOs with integrated embA); W, wild-type genomic DNA.

Fig. 2.

Arabinosyltransferase activity assays. Extracts from M. smegmatis strains were prepared and used in an in vitro assay for arabinosyltransferase activity. (a) TLC and (b) Dionex HPAEC was used to analyse the product formed after incubation with the pentasaccharide acceptor. (a) TLC of M. smegmatis strains. Wild-type (lanes 1 and 2), ΔembA (3 and 4), ΔembA complemented with embA_Msm (5 and 6) and ΔembA complemented with embA_Mtb (7 and 8). Lanes 1, 3, 5 and 7 are reactions with the addition of an acceptor pentasaccharide; lanes 2, 4, 6 and 8 are reactions with no acceptor. The acceptor pentasaccharide (lane 9) was visualized on TLC with α-naphthol. (b) The enzymically formed radioactive product was purified from the TLC plate and subjected to endoarabinanase digestion followed by Dionex HPAEC analysis, resulting in a single Ara₆ peak. (c) ¹⁴C-labelled AG digested with endoarabinanase showing three peaks as Ara₂, Ara₆ and cyclic Gal₄, as established by Xin et al. (1997).

Fig. 3.

Promoter activity of the embA upstream region. (a) Sequence of the embCA intergenic region carrying P_embA(Mtb). The stop and start codons of embC and embA are underlined; the predicted −10 region is in large text. The bases in bold were mutated to Gs in plasmid pEMBA-M. (b) Promoter activity from P_embA(Mtb) in M. smegmatis and M. tuberculosis. M. smegmatis transformants were grown in 10 ml 7H9-OADC for 30 h. M. tuberculosis transformants were grown in 10 ml stationary cultures of 7H9-OADC-Tw for 14 days. Results are the means±sd of three individual transformants each assayed in duplicate and are expressed as Miller units. (c) RT-PCR analysis. Agarose gel analysis of RT-PCR on the embC–embA and embA–embB junctions of the embCAB locus. PCR with primers EMBCF2 and EMBAR failed to produce the expected 0.795 kb product (lane 1) while the primer pair EMBAF1 and EMBBR produced the expected 0.649 kb product (lane 2). Lane M is a 1 kb DNA ladder. (d) Genetic organization of the emb region on the M. tuberculosis chromosome and Northern analysis of total RNA from M. tuberculosis hybridized to labelled embA.

Fig. 4.

P_embA(Mtb) activity during aerobic growth and under hypoxic conditions. (a) Aerobic growth. M. tuberculosis was grown in 10 ml static cultures of 7H9-OADC-Tw (open symbols) or on 7H10-OADC plates (filled symbols). (b) M. smegmatis was grown in 10 ml 7H9-OADC. (c) Hypoxic growth. P_embA(Mtb) activity in hypoxic conditions (white columns) is compared to aerobic conditions (black columns). M. tuberculosis was grown under hypoxic conditions for 3 weeks and M. smegmatis for 2 weeks. Results are the means±sd of three individual transformants each assayed in duplicate and are expressed as Miller units.